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Methods

C57Bl/6 J mice underwent intraperitoneal inoculation with scrapie brain homogenates
and were later induced with experimental autoimmune encephalomyelitis by inoculation
of MOG35-55 in complete Freund's adjuvant supplemented with pertussis toxin. Spleen and lymph
node cells from the co-induced animals were reactivated and subsequently injected
into naïve mice as viable cells or as cell homogenates. Control groups were infected
with viable and homogenized scrapie immune cells only with complete Freund's adjuvant.
Prion disease incubation times as well as levels and sites of PrPSc deposition were next evaluated.

Results

We first show that acceleration of prion disease by experimental autoimmune encephalomyelitis
requires the presence of high levels of spleen PrPSc. Next, we present evidence that mice infected with activated prion-experimental autoimmune
encephalomyelitis viable cells succumb to prion disease considerably faster than do
mice infected with equivalent cell extracts or other controls, concomitant with the
deposition of PrPSc aggregates in white matter areas in brains and spinal cords.

Conclusions

Our results indicate that inflammatory targeting of viable prion-infected immune cells
to the central nervous system accelerates prion disease propagation. We also show
that in the absence of such targeting it is the load of PrPSc in the inoculum that determines the infectivity titers for subsequent transmissions.
Both of these conclusions have important clinical implications as related to the risk
of prion disease contamination of blood products.

Keywords:

Brain; EAE; immune cells; infiltrates; prion; PrPSc; spinal cord

Background

Prion diseases are a group of fatal neurodegenerative disorders that include Creutzfeldt-Jakob
disease and kuru in humans, bovine spongiform encephalopathy in cattle, scrapie in
sheep and goats, and chronic wasting disease in deer [1]. This group of diseases is caused by the accumulation of a misfolded and oxidized
isoform of PrPSc, a normal membrane protein believed to play a role in the protection against oxidative
insults [2,3]. All forms of prion diseases are characterized by long incubation periods, which
in humans can sometimes amount to decades [3-6]. Although the exact mechanism of prion propagation is unknown, a general sequence
of events has been outlined that is consistent with most of the available data [4]. First, and regardless of the route of infection, prions replicate in lymphoid organs,
as shown by the fact that both infectivity and accumulation of PrPSc are initially detected in the spleens of the infected animals [5-8]. Prions invade the nervous system by a mechanism believed to involve transmigration
of infected lymphoid cells as well as retrograde transport in ascending peripheral
neural tracts [4,9-12]. Once prions enter the brain, prion replication and PrPSc accumulation seems to occur faster than in the lymphoid system, leading to the certain
death of the affected individuals [13-15]. In the presence of inflammatory conditions affecting peripheral organs, activated
lymphoreticular cells induce deposition of PrPSc and prion infectivity in the sites of infiltration in prion-infected animals [16]. For example, mastitis in sheep results in deposition of PrPSc in the inflamed mammary glands [17].

While no differences in prion disease incubation time or clinical symptoms were reported
in these peripheral infection cases, mice incubating scrapie and induced for experimental
autoimmune encephalomyelitis (EAE), an autoimmune inflammatory disease of the central
nervous system (CNS) [18,19], died from a progressive neurological disease long before the control mice succumbed
to classical scrapie [20]. Surprisingly, mice affected by the scrapie-EAE syndrome showed almost undetectable
to high levels of brain PrPSc, demonstrating that there is no correlation between the clinical status of the affected
scrapie-EAE mouse and PrPSc accumulation. Immunostaining studies revealed PrPSc depositions in the demyelinated white matter spinal cord areas of the co-induced mice,
mostly co-localized with hematopoietic cell infiltrates. In classical scrapie, PrPSc aggregates are mostly found in the gray matter [21].

In this project we investigated the mechanism of prion disease acceleration by CNS
inflammation. In particular, we asked whether accelerated prion disease in the co-induced
animals requires the targeted delivery of PrPSc to the CNS by activated immune cells. To this aim, we compared the effect of EAE induction
on prion disease kinetics at several time points before and after infection with scrapie
and found that acceleration of disease is the strongest at one month after prion infection,
when PrPSc is largely accumulated in immune organs such as the spleen [22]. Next, we inoculated naïve mice with viable or homogenized activated immune cells
collected from scrapie-EAE mice and controls. Only the activated viable cells generated
CNS inflammation and accelerated prion disease, as compared with non-specific immune
activation, or with scrapie brain homogenates with similar PrPSc loads. Our results therefore suggest that EAE-dependent acceleration of fatal prion
disease results from the infiltration of PrPSc loaded immune cells into the CNS. Additional results presented here indicate that,
in the absence of such infiltration, incubation times of prion disease relate mostly
to the levels of the inoculated PrPSc.

Methods

Animals

Female C57Bl/6 J mice were purchased from Harlan (Hebrew University, Jerusalem, Israel)
and housed in the animal care facility in compliance with the standard guidelines
for animal care. All experiments were approved by the Institutional Animal Care and
Use Committee.

Prion infection

Four- to five-week-old mice were inoculated by intraperitoneal injection with 100
μL or intracerebral injection with 50 μL of 1% scrapie brain homogenate. Mice were
followed closely for disease signs until disease manifestation and killed when too
sick to reach food and water.

Experimental autoimmune encephalomyelitis induction

Five- to eight-week-old female C57BL/6 J mice were immunized with an emulsion containing
300 μg of MOG35-55 (70% purified; synthesized at the Hebrew University, Jerusalem, Israel) solubilized
in saline and an equal volume of complete Freund's adjuvant (CFA; Sigma, Rehovot,
Israel) supplemented with 5 mg/mL of heat-killed mycobacteria Tuberculosis H37RA (Difco Laboratories, Detroit, MI, USA). The inoculum (0.2 mL) was injected subcutaneously
in both flanks. On the day of inoculation and 48 hours later, 100 ng of pertussis
toxin (List Biological Laboratories, Inc. Cambell, CA, USA) in 0.1 mL saline was also
administered by intraperitoneal injection. Control groups were inoculated the same
way but without MOG35-55.

Passive transfer of experimental autoimmune encephalomyelitis

Nine days post induction mice were killed and cells were obtained from the spleen
and lymph nodes. Cells were suspended in Roswell Park Memorial Institute medium (Biological
Industries, Beit Haemek, Israel) supplemented with 10% fetal calf serum, 1 mM L-glutamine,
antibiotics, MOG35-55 (20 μg/mL) and mouse recombinant IL-2 (50 units/mL; Peprotech, Rocky Hill, NJ, USA)
and incubated for three days in a CO2 humidified incubator. Cells were harvested and live cells were separated with Ficoll-Paque
(GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and injected into naïve mice. In
control groups of cells extracts, cells were frozen after separation, thawed three
times and then injected into naïve mice. On the day of inoculation and 48 hours later,
100 ng of pertussis toxin in 0.1 mL saline was also administered by intraperitoneal
injection. Mice were followed for clinical symptoms of EAE and evaluated according
to a 0 to 6 point score (0 represents no clinical signs and 6 represents death as
a result of disease).

Pathological examinations

Histological evaluations were performed on paraffin-embedded sections of brain and
spinal cord samples. Sections were stained with luxol fast blue/Periodic acid Schiff
(PAS) to assess demyelination. Consecutive sections were used for immunohistochemistry
with antibodies against the following targets: macrophages and activated microglia
(anti-MAC3; BD Pharmingen, San Diego, CA, USA); T-cells (anti-CD3; Serotec, Oxford,
UK); and prion protein (anti-PrP 6H4; Prionics).

Statistical analysis

The clinical scores of disease in groups of animals with EAE and EAE + scrapie were
compared using Kruskal-Wallis one-way analysis of variance on ranks. The survival
curves were compared using chi-squared test.

Results and discussion

Regardless of the route of prion infection, PrPSc is first accumulated in immune organs such as the spleen and lymph nodes and appears
in the infected brains much later in the incubation period [22,23]. In the scrapie RML model (mouse), it takes one month after intracerebral or intraperitoneal
infection for PrPSc to accumulate in the spleen at high levels. Indeed, Figure 1A shows that while no PrPSc was detected in the brain one month after intraperitoneal infection with a scrapie
brain homogenate, PrPSc was easily detectable in the spleen at this time point. To test whether the presence
of PrPSc in immune organs is required for disease acceleration by CNS inflammation, we induced
MOG-EAE both one week and one month after scrapie infection (intracerebral injection)
of C57Bl/6 J female mice. Animals were followed for clinical symptoms of both EAE
and scrapie (Figure 1C). Figure 1B shows no difference in the severity of EAE signs, such as paralysis of the lower
limbs, during the first 30 days in the groups induced for EAE after prion infection
as compared to control EAE groups [24]. While most mice recovered from the acute disease at this point, the group induced
for EAE one month after scrapie infection continued to deteriorate, demonstrating
new neurological signs such as tail rigidity, kyphosis, ataxia, tremor and bradykinesia,
which are classical characteristics of prion disease [25]. Similar signs were observed at 80 days or later in the group induced for EAE one
week after scrapie infection. Figure 1D depicts the survival of mice from these groups as compared to control scrapie-infected
mice. Indeed, while mice induced for EAE one week after prion infection showed some
acceleration of disease progression (median: 155 days) as compared to scrapie controls
(median: 175 days), a much larger difference in survival was observed between the
scrapie control (median: 175 days) and the group also induced for EAE at one month
of prion infection (median: 60 days). Only a marginal acceleration of disease was
observed when EAE was induced in the mice before scrapie infection (not shown). These
results indicate that the rate of prion disease acceleration by CNS inflammation may
depend on the levels of PrPSc accumulating in lymphoid organs at the different time points.

Figure 1.PrPSc in spleen is required for the acceleration of prion disease by experimental autoimmune
encephalomyelitis. (A) Brain and spleen samples from scrapie-infected mice (C57Bl/6 J, RML strain) at several
incubation points (1 week, 1 month, sick) were tested for the levels of proteinase
K digested PrPSc. (B) Prion-infected mice were induced for MOG-EAE one week (n = 5) and one month (n = 6)
after prion infection and followed for signs of disease. The clinical scores of the
group induced with EAE one month post infection were significantly different (P = 0.025) as compared to naïve mice induced for EAE (n = 6). The clinical scores of
mice with EAE induced one week post infection were not statistically different (P = 0.5) as compared to naïve mice induced for EAE (n = 6). (C) Table presenting chronological symptoms of the scrapie-EAE syndrome. (D) Survival curves of mice from the designated groups. Scrapie/EAE at one month was statistically
different (P = 0.0033) as compared to the corresponding scrapie/EAE at one week.

We next tested the incubation time resulting from prion infection of naïve mice either
with viable or with homogenized scrapie-EAE immune cells. To this effect, C57Bl/6
J mice were infected intraperitoneally with scrapie RML prions and one month later
induced either for EAE (MOG (myelin oligodendrocyte glycoprotein) + CFA + pertussis
toxin) or for adjuvant only inflammation (CFA + pertussis toxin). Nine days thereafter,
scrapie-EAE mice and controls were killed, their spleens and lymph nodes harvested
and reactivated in vitro with MOG and subsequently enriched for viable cells only (see Methods). An outline
for these experiments is depicted in Figure 2. Before inoculation into naïve mice groups, cell fractions were tested for PrPSc levels as compared to sequential dilutions of prion-infected brain and spleen samples
(Figure 3A). Next, groups of naïve mice (four to five animals in each of two combined experiments)
were inoculated intraperitoneally with the viable cells or with their homogenates,
as well as with two dilutions (10-2 and 10-4) of scrapie-infected brain homogenates. Each mouse was infected with the equivalent
of cells per homogenate harvested from two donor mice, comprising a PrPSc load comparable to that of a 10-4 dilution of scrapie-infected brains. All groups of infected mice were followed closely
for signs of neurological disease. Mild EAE signs, such as limp tail and hind limb
weakness (paraparesis), were observed only in the mice inoculated with scrapie-EAE
viable cells (Figure 3B), starting at day 30 post infection. Figure 3C presents the survival data for the mice in the different experimental groups. Mice
from the groups infected with CFA only-induced cells, as well as with cell homogenates
and 10-4 brain dilutions, developed fatal prion disease at about 220 days, indicating no difference
in the PrPSc to infectivity ratio of the homogenates from cells and brain, as well as for viable
cells induced only with CFA. Contrarily, the group infected with viable scrapie-EAE
cells succumbed to prion disease 30 days earlier, even before the mice infected with
a 10-2 scrapie brain dilution. These results indicate that the PrPSc to infectivity ratio is at least 100 times lower in the activated viable cells, consistent
with the possibility that accelerated prion disease is caused by the inflammation-dependent
targeting of prions into the CNS.

Figure 2.Outline of scrapie-experimental autoimmune encephalomyelitis transfer experiments. C57Bl/6 J mice were induced for either MOG-EAE or CFA disease one month after intraperitoneal
infection with scrapie RML. Nine days later mice were killed and immune cells collected
and incubated with MOG for three days, before their inoculation as viable or homogenate
samples into naïve mice. Control scrapie-infected brain samples were also inoculated
in naïve mice.

Figure 3.Infection of mice with viable scrapie-experimental autoimmune encephalomyelitis immune
cells shortened the prion disease incubation period. (A) PrPSc levels in brains and immune samples. (B) EAE clinical scores in mice inoculated with viable or homogenated cells after activation
ex vivo for three days with MOG. (C) Survival curves of mice infected with the designated inocula. The survival curve of
group 1 was significantly different (P < 0.05) as compared to survival curves of groups 2,3,4 and 6. Results represent eight
to ten mice in each category.

PrPSc in white matter demyelinated areas

The pathological examination of scrapie-infected mice induced for EAE [20] revealed depositions of PrPSc in white matter inflamed areas. Figure 4 shows the differences in demyelination and PrPSc deposition in the white matter spinal cords of naïve mice infected with viable as
compared to homogenized scrapie-EAE cells. Indeed, spinal cords of mice infected with
viable scrapie-EAE cells present marked subpial demyelination co-localized with activated
microglia, macrophages and moderate levels of PrPSc accumulation, mainly at the border of the lesion (Figure 4). Due to the long time lapse from infection with the viable cells to prion disease
manifestation, it is hard to determine whether prion-infected macrophages were the
ones that deposited the PrPSc in the demyelinated spinal cord, or whether their presence represents the engulfing
of other prion-infected cells, such as T-cells. Such a co-localization between PrPSc, Mac-3 and demyelination was not present in the spinal cords of mice infected with
cells homogenates. Extensive PrPSc deposition was also observed in brain white matter areas such as the commissura anterior
of mice infected with scrapie-EAE viable cells (Figure 5), again contrary to the low levels of PrPSc observed in the brain white matter of mice infected with cell homogenates. These results
are again consistent with the idea that viable prion-infected immune cells activated
against myelin components, such as MOG in this case, may carry PrPSc into white matter CNS areas, a feature not frequently seen in prion infection [21]. For unknown reasons, this route of infection is more virulent, as compared to classic
prion neuroinvasion, and is believed to involve retrograde transport of prions to
the brain. Both routes of neuroinvasion may occur in parallel and produce additive
signs of disease.

Figure 4.Pathology spinal cords. Upper panel: viable cells. Pathology is characterized by marked subpial demyelination
(A, B, Luxol fast blue). The rectangle in (A) marks the region enlarged in (B-D).
The demyelinated areas show some activated microglia and macrophages (C, Mac-3) as
well as moderate PrPSc deposition, which is most abundant at the lesion border (D, arrowheads, PrPSc). Lower panel: homogenated cells. In contrast, control animals were devoid of demyelination
(E, F, luxol fast blue). The rectangle in (E) marks the region enlarged in (F-H).
White matter only reveals some activated microglia (G, Mac-3) but no PrPSc deposition (H).

We have shown in Figure 3 that while most of the inoculated samples comprised similar levels of PrPSc, the sample consisting of viable scrapie-EAE cells generated disease in the naïve
mice significantly earlier than all others, indicating that levels of PrPSc are not the only designator of prion disease incubation time. In our previous work
[20], we showed that mice may succumb to the co-induced scrapie-EAE disease at similar
incubation times with very different levels of brain PrPSc, those varying from undetectable levels to the high levels observed in mice with
classical scrapie. To this effect, we next investigated whether mice succumbing to
the co-induced disease generate a new prion strain that can transmit disease independent
of PrPSc inoculum levels, or whether disease transmission from these mice correlate with the
accumulated PrPSc levels when in brain homogenates.

To separate these possibilities, we infected groups of 5 C57Bl/6 J mice with the brain
homogenates of the samples described in Figure 6. Group 1 was inoculated with the brain homogenate from a normal mouse induced for
EAE, group 2 was infected with the homogenate of a still healthy mouse incubating
classical scrapie, and group 3 was infected with the homogenate of a scrapie sick
mouse. Groups 4 to 6 were infected with brains of mice that succumbed to the scrapie-EAE
co-induced disease at a similar time point; however, each with a different level of
brain PrPSc. Figure 6 shows that samples 1, 2 and 4 could not transmit disease to naïve mice for at least
300 days post infection. This is specifically surprising for sample 4, generated from
the brain of a mouse that succumbed to the co-induced disease. Sample 5, which presents
marginal levels of PrPSc, generated intermediate levels of infectivity in the recipient group. Only mice inoculated
with samples 3 and 6 (high levels of PrPSc) succumbed to prion disease, at short incubation times. These results indicate that
the scrapie-EAE syndrome, while causing accelerated disease, does not induce the replication
and accumulation of prions that can further transmit disease in the absence of PrPSc. These results are therefore consistent with the notion that inflammation-dependent
targeting of PrPSc can accelerate disease regardless of the inoculated PrPSc levels. In the absence of such PrPSc targeting, as is the case for the total brain homogenates in these second transmission
experiments, it is the levels of PrPSc that determine the incubation time of disease manifestation.

Figure 6.Secondary transmission of co-induced brain homogenates depends on PrPSc levels. (A) Sample legend. (B) Immunoblots presenting levels of PrPSc in the samples described in A. (C) Survival curves of mice (n = 5 for each group) infected with the samples described
in A and B. The survival curve of scrapie-EAE (PrPSc high) was significantly different (P < 0.02) as compared to scrapie-EAE (PrPSc low).

Conclusions

We have shown in this work that acceleration of prion disease by CNS inflammation,
as is the case for EAE induction, requires the presence of substantial levels of PrPSc in the immune cells of prion-infected mice. Such PrPSc-rich cells may then be targeted by CNS inflammation for infiltration into the CNS,
as demonstrated by the presence of PrPSc deposits in white mater areas of brains and spinal cords. This was true for mice induced
with EAE a month after scrapie infection [20] as well as for mice infected directly with viable and activated scrapie-EAE cells
(Figures 3 and 4). Our experiments also show that the inflammation-mediated disease does not constitute
a more virulent prion strain, since secondary transmission from scrapie-EAE mouse
homogenized brain samples indicated that prion titers were dependent mostly on the
inoculated levels of PrPSc. These results are consistent with previous observations that suggest inflammation
may affect the sites of PrPSc accumulation [16,17] and, in the case of CNS inflammation, accelerate the presentation of fatal disease
[20], sometimes in the absence of high levels of PrPSc in the brains of the affected animals. It is possible that low levels of prions that
have infiltrated into white matter areas are more toxic and facilitate the propagation
of disease in a more efficient way. Both mechanisms, retrograde transport of prions
and inflammation-dependent neuroinvasion, may occur distinctively or in parallel,
explaining how similarly sick mice present different levels of brain PrPSc at the end point of disease. It is also possible that a variable balance between EAE-linked
and scrapie symptoms can account for this phenomenon.

The fact that incubation times for second-generation transmission of disease from
homogenates of scrapie-EAE brains were independent of the clinical status of donor
mice and related only to PrPSc levels indicates that inflammation did not change the virulence properties of the
initial prion strain, but rather accelerated the targeting of the infectious agent
to vital areas, resulting in a shorter disease. This may explain why endogenous blood
prions carry more infectivity than predicted from their low blood PrPSc content [4,26,27]. Indeed, our results suggest that infection by viable cells carrying PrPSc may be more rapid and that the infectivity of a specific blood sample may depend not
only on the prion incubation status of the blood donor, but also on the inflammatory
state of both the donor and recipient on the day of the transfusion.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

YF-L made substantial contributions to the conception and design, acquisition of data,
analysis and interpretation of results and drafting of the manuscript. RH made substantial
contributions to the acquisition of data and its interpretation. HB made substantial
contributions to the acquisition of data, interpretation of data and critical reading
of the manuscript. TM-S made substantial contributions to the acquisition of data
and interpretation of results. OA made substantial contributions to the interpretation
of data and critical reading of the manuscript. HO made substantial contributions
to the conception and design, acquisition of data, and interpretation of results and
critical reading of the manuscript. RG made substantial contributions to the conception
and design, acquisition of data, interpretation of results, drafting the manuscript,
and has given final approval of the version to be published. All authors read an approved
the final manuscript.

Acknowledgements

This work was funded by the Morasha ISF foundation and by the Agnes Ginges foundation.